When bone is subjected to overloading or fatigue, its apparent elastic modulus decreases. The decreased modulus is the macroscopic manifestation of microdamage, microcracks, and, in cancellous bone, the fracture of trabeculae. The resulting localized decreases in the apparent level stiffness may result in an increased fracture risk due to redistribution of loads. From a biological adaptation standpoint, damage with the bone tissue is believed to stimulate remodeling. As such, damage development in bone has implications for both whole bone strength and bone adaptation. Current methods of assessing the presence of microdamage are limited to two-dimensional imaging of thin sections, and provide minimal information regarding the three-dimensional distribution of damage or orientation of cracks. However, the effects of damage on bone strength and stiffness cannot be fully assessed without this information. It has been shown that the modulus reduction of cortical bone differs for different modes of damaging loads. Similar findings have been reported for cancellous bone. These differences are likely due to different damage patterns, or localization of damage. Thus, the ability to quantify the three-dimensional distribution and location of damage in bone will provide insight into the concomitant macroscopic property changes. The goal of this study is to develop a methodology for three-dimensional detection and visualization of microdamage and microcracks in bone specimens in vitro using micro-computed tomography (muCT). A radio-opaque marker will be developed which preferentially attaches to damaged regions of bone by chelating to exposed calcium (e.g. on the new surfaces created by microcracks). The marker will appear as regions of increased density in muCT, allowing visualization of the damage pattern in three dimensions. Specifically, the aims of the study are to: 1. Identify one or more metal chelating agents having two or more carboxy functional groups (multi-functional carboxylic acids or proteins) that will chelate with both calcium in bone and one or more heavy metal ions which act as a radio-opaque markers. 2. Determine the optimum stain chemistry (chelating agent and metal, concentrations, pH), staining technique, and exposure time for labeling microdamage in bone. 3. Identify the optimal scanning parameters for imaging microdamage in bone using micro-computed tomography (muCT) for each variant in the stain chemistry and staining technique. 4. Image the three-dimensional damage developed in cortical bone specimens following low cycle fatigue in four point bending. Differences in damage patterns due to pure bending and combined bending and shear loading will be identified.